Evaluation of the protection exerted by Pisum sativum Ferredoxin-NADP(H) Reductase against injury induced by hypothermia on Cos-7 cells.

Hypothermia is employed as a method to diminish metabolism rates and preserve tissues and cells. However, low temperatures constitute a stress that produces biochemical changes whose extension depends on the duration and degree of cold exposure and is manifested when physiological temperature is restored. For many cellular types, cold induces an oxidative stress that is dependent on the elevation of intracellular iron, damages macromolecules, and is prevented by the addition of iron chelators. Pisum sativum Ferredoxin-NADP(H) Reductase (FNR) has been implicated in protection from injury mediated by intracellular iron increase and successfully used to reduce oxidative damage on bacterial, plant and mammalian systems. In this work, FNR was expressed in Cos-7 cells; then, they were submitted to cold incubation and iron overload to ascertain whether this enzyme was capable of diminishing the harm produced by these challenges. Contrary to expected, FNR was not protective and even exacerbated the damage under certain circumstances. It was also found that the injury induced by hypothermia in Cos-7 cells presented both iron-dependent and iron-independent components of damage when cells were actively dividing but only iron-independent component when cells were in an arrested state. This is in agreement with previous findings which showed that iron-dependent damage is also an energy-dependent process.

Expression and distribution of aquaporin 8 in rat hepatocytes cold stored 72 hours in modified University of Wisconsin and bes-gluconate-sucrose solutions. Study of their correlation with water content.

Since few data are availble on the genetic responses to low temperatures, we investigated if cold storage of hepatocytes (0 degree C, mUW or BGS solutions, 72 h) can affect gene expression and/or cellular localization of AQP8 and their correlation with water movements. Cold preserved hepatocytes showed a significant decrease in water content (P less than 0.05) but were able to regulate their volume when they returned to physiological conditions. These changes were not related to modulation in the expression and the pattern of distribution of AQP8 suggesting that other mechanisms are involved. The study of the quantitative changes in the expression of genes coding for liver specific proteins in cold preserved hepatic cells is of interest in order to develop new preservation methods or solutions that could contribute to maintain the utility of these cells when destined to be applied in clinical models.

Hypothermic storage of periportal and perivenous rat hepatocytes.

High yields of intact parenchymal cells are produced by the two-step Digitonin-collagenase perfusion of whole liver, and it has gained wide acceptance for biochemical and cellular analyses of zonal hepatocytes. The development reached by this methodology is in contrast to the time-limited use of the isolated cells unless those other methods, such as primary cultures, are employed. An alternative option to have cells ready to be used for several days, is the cold storage in University of Wisconsin solution as a preservation solution. This procedure is easy, not too expensive, and does not require specialized equipment. We study the competence of this system to maintain liver cells: mixed or total cells and cell-enriched fractions. We affirm viability of hepatocytes during hypothermic storage (UW-96 h-4 degrees C) by Trypan Blue exclusion, the capacity to retain cytoplasmic enzymes, metabolic competence to maintain total Glutathione content, and immunocytochemistry (gene detection). After 96 h of cold storage, mixed cells and cell-enriched fractions, were submitted to normothermic incubation (120 min, 37 degrees C) and we check Trypan Blue exclusion, cytoplasmic enzyme release, and the capacity of cell populations to synthesize urea. The results show that it is possible to use, after several days of storage, mixed liver cells and cell-enriched fractions in metabolic and gene expression studies. This procedure allows us to reduce the number of experimental animals needed, to save experimental time and costs, and to facilitate further studies in vitro about the basis and consequences of metabolic heterogeneity of the liver cell plate.

Glutathione movements during cold preservation of rat hepatocytes.

In this study we have examined the movements of glutathione (GSH) during cold preservation of rat hepatocytes in University of Wisconsin solution. During the preservation process at a low temperature (4 degrees C), with a high extracellular potassium concentration, an extracellular nondiffusible anion (lactobionate), and a Cl(-)-free medium, there is a depletion of metabolites and the development of a time-dependent injury. Also, there is a loss of GSH that is not compensated by transport or synthesis and is basically due to increased catabolic processes. This sensitizes the cells to different forms of oxidative injury, which can play a negative role during transplantation. The addition of GSH improves liver cell preservation but the mechanism is unclear. To elucidate this process we have isolated hepatocytes and preserved them under different conditions: with or without GSH: in the presence of DL-buthionine-[S,R]-sulfoximine, an inhibitor of glutathione synthetase, and acivicine to inhibit the ectoactivity of cellular gammaglutamyl transpeptidase; or by obtaining hepatocytes from rats depleted of GSH by an injection of diethyl maleate. Under all these conditions we evaluated the GSH content of the cells during cold storage. We also report the time course of accumulation of [glycine-2-3H]GSH. Our results show that during hypothermic storage in University of Wisconsin solution, hepatocytes are permeable to GSH, and the mechanism involved is a rapid nonsaturable process, with linear dependence of the extracellular GSH concentration. This finding may have valuable applications in the improvement of the delivery of compounds to cells.